Proteases of animal, vegetable or microbial origin are commonly used with advantage in a variety of applications such as detergents, pharmaceutical composition. e.g. digestants, antiinflammatory drugs, etc., cosmetics, meat tenderizing agents, silk scouring agents, beer production and so on.
However, it has been pointed out that being proteins heterogenous to man, these proteases in detergents, cosmetic and pharmaceutical products, etc. present antigenicity and dermal hypersensitivity problems and at times elicit intense irritable responses depending on individuals. A further problem with these enzymes is that they are relatively labile.
Particularly in media rich in water or in aqueous solutions, proteases are not only denatured but also undergo autolysis. It is also known that storage at ambient temperature results in rapid inactivation. Therefore, it has heretofore been difficult to supply the consumer with stable protease-containing products.
In order to solve the antigenicity and other safety problems with this type of enzyme, namely for suppressing the antigenicity and prolonging the blood half-life of the enzyme in systemic regimens for the treatment of diseases, it has been proposed to modify uricase or asparaginase with polyethylene glycol (Japanese Patent Publication No. 61-42558) or modify streptokinase with polyethylene glycol (Japanese Kokai Patent Publication No. 57-118789). As an approach to the stability problem of proteases, it has been shown with chymotrypsin and certain other enzymes that a chemical modification contributory to intramolecular bridging is effective (Biochimica et Biophysica Acta 522, 277-283, 1987 and 485, 1-12, 1977. Furthermore, it has been demonstrated that the binding of a water-soluble polymer such as a polysaccharide, polyethylene glycol, a protein or the like to a manganese superoxide dismutase results in suppression of the antigenicity and improvement of the thermal stability of the enzyme (Japanese Kokai Patent Publication No. 58-16685).
However, there is not known an implementation technology which would contribute to reduction of the antigenicity and skin sensitization potentials and, at the same time, to the stability of the enzyme. Since dermal hypersensitivity, inter alia, is a very delicate reaction, the inhibition of this reaction is technically a very difficult proposition. Moreover, since the substrate of proteases are generally high molecular weight substances, their enzymatic activity and their thermal stability are seriously sacrificed by such modification depending on the degree of modification. Therefore, any modification of the enzymes is also very difficult.
Therefore, the inventors of the present invention explored into this realm of technology with a view to improving the stability of proteases without sacrificing their activity in order that those enzymes may be better exploited in a broad spectrum of uses such as detergents, cosmetics, drugs and so forth. The above exploration and subsequent research resulted in accomplishment of the present invention.
The present invention is therefore directed to a modified protease with reduced skin sensitization and antigenic potentials, improved stability and high activity, a method of producing such modified protease, and a cosmetic composition containing the modified protease.